1. Field of the Invention
This invention relates to an immunoassay for detecting or quantifying core proteins of the structural region of Non-A,Non-B hepatitis virus in specimens, to monoclonal antibodies for use in the immunoassay, to hybridomas capable of producing the monoclonal antibodies, and to a test kit comprising the monoclonal antibodies.
2. Prior Art
Post-transfusion hepatitis refers literally to a hepatitis caused by transfusion. As a causative virus of the post-transfusion hepatitis, hepatitis B virus (i.e., HBV) was first identified, but it was suspected thereafter that an other virus was involved in the post-transfusion hepatitis in addition to HBV. This virus that is different from HBV was named Non-A,Non-B hapatitis virus (hereinafter, referred to as "NANBV") or hepatitis C virus (i.e., HCV), and its identification was demanded .
Non-A,Non-B hepatitis corresponds to at least 90% of the post-transfusion hepatitis, and 50-80% of the infected patients indicate chronic symptoms which subsequently transfer to cirrhosis then hepatoma in high proportions, and because of this, there has been a great demand on physiological functions of this disease. The Non-A,Non-B hapatitis has been clinically identified mainly by the so-called exclusion-diagnosis method in which it is first confirmed whether this disease is hapatitis A or hepatitis B or other hepatitis caused by known viruses (e.g., cytomegalovirus and Epstein-Barr virus) by which liver disorders are caused, after which if the disease is other than the known hepatitis then it is determined as being Non-A,Non-B hepatitis.
The cloning of a gene of the virus in question was thereafter carried out by a group of Choo et al. using NANBV-infected chimpanzee plasmas (see Science 244:359-362, 1989), and a method for the diagnosis of the Non-A,Non-B hapatitis was developed in which the detection of antibodies raised against NANBV was carried out by use of recombinant antigens prepared by introducing parts of the non-structural region of NANBV genes (i.e., extending from NS3 to NS4) (see Science 244:362-364, 1989; and JP-A-2-500880).
The commercially available first generation reagent that contains a recombinant antigen (i.e., c100-3 protein) developed by Chiron Corp. (USA) can detect as positive 70-80% of patients with chronic Non-A,Non-B hapatitis, but because the c100-3 antibody titer does not rise at the early infection and the detection of the antibody to c100-3 is impossible, it was known that there were cases undetected even when infected and that considerably non-specific responses were observed in sera from patients with autoimmune diseases or hyper-.gamma. globulin sera.
More recently, the second generation regents containing an additional core antigen which is a structural protein of NANBV were developed whereby about 90% of patients infected with NANBV could be detected, but the detection of patients with sporadic Non-A,Non-B hepatitis is as low as about 40%. In addition to the above-mentioned antibody-detection methods, developed are methods for the detection of NANBV genes in which the presence or absence of the NANBV genes is determined by PCR (see Science 230:1350-1354, 1985) or DNA probe method.
However, there are some difficult problems in the detection methods using the PCR method. As such problems, the following are exemplified: reverse transcription is required because NANBV is a RNA virus; the loss of cDNA occurs during the reverse transcription; specific amplification equipments are needed; it is impossible to treat a lot of samples at one time because the operation is complex; and contamination of samples are often observed.
Regarding Non-A, Non-B hapatitis virus, its viral level is very low in patient bodies and its in vitro proliferation system has not been established, so no experimental immunological sera have been obtained using native NANBV virions or purified NANBV proteins. Human sera include different productions of antibodies to NANBV-related antigens depending on individuals, namely, individuals showing a high antibody titer, individuals not capable of eliciting the antibodies, and individuals having an antibody to an antigen present in a certain region of NANBV but not any antibodies to antigens in other regions. Additionally, because of polyclonal antibodies, human sera include antibodies to foreign substances other than NANBV, so estimation of obtained results has to be conducted while taking undesirable cross-reactions into consideration.
When unknown antigens are estimated by use of antibodies, well attention is required for the above reasons. In this context, the development of a method of the detection of NANBV itself for definitive diagnosis has been noted.
With respect to monoclonal antibodies to NANBV, prior arts do not describe any practical production of such monoclonal antibodies although its possibility has been suggested (see EP-B-318,216 and EP-B-388,232), except for monoclonal antibodies specific for epitopes on core proteins of NANBV (see JP-A-5-260960). But the latter monoclonal antibodies differ from the monoclonal antibodies of this invention in recognizable epitope types, assay methods, sensitivities for detection, preparation methods, etc.
A high degree of mutation are observed in a Non-A,Non-B hepatitis virus genome as other class of RNA viruses. As stated above, the level of NANBV virions themselves are very low in patients infected with NANBV and thus the level of antibodies to NANBV-related antigens in the patients' sera is low, so all the patients can not be detected using reagents for detection of the antibodies. Because Non-A,Non-B hepatitis whose prevention has not yet been achieved is assumed to transfer to cirrhosis and hepatoma up to 10-30 years after infection in many infected persons (including carriers), early finding and early treatment are of course very important. Recently, it has been reported that the interferon (referred to as "IFN", hereinafter) therapy is effective for chronic patients, but the liver disorders can not be completely cured by the INF treatment alone, as seen from the fact that there are many cases re-suffering from the hepatitis even when the IFN treatment was terminated. Thus complete exclusion of NANBV from the patient bodies is difficult. It is rather significant to grasp the pathology of the disease in order to assume the treatment or prognosis, and because of this, it is strongly desired not only to detect antibodies to NANBV-related antigens but also to measure NANBV-related markers (i.e., antigens). Thus, the development of a method of obtaining NANBV virions, particularly NANBV-related antigens from infected sera in a simple way and in a high yield, as well as the detection or quantitative analysis of the virions or antigens, has been demanded.